Nucleotides and nucleotide sugars are released in regulated fashion from both excitable and non- excitable cells and act as extracellular signaling molecules to regulate a remarkably broad array of physiological responses ranging from neurotransmission to epithelial ion transport to platelet aggregation. At least fifteen different P2 receptors recognize extracellular nucleotides as their cognate agonists, and eight of these comprise the family of metabotropic G protein-coupled P2Y receptors. Although signals emanating from P2Y receptors convey important physiological and pathophysiological responses in essentially all tissues, physiological and molecular understanding of the action of these receptors has lagged, largely due to lack of reliable molecules to probe unambiguously the actions of single receptor subtypes. An antagonist (clopidogrel;Plavix) of the platelet ADP-activated P2Y12 receptor has made an enormous impact in modern therapeutics, and the potential for drugs that clinically target other G protein-coupled P2Y receptors is both very large and as yet unrealized. Our research program has developed and applied the first selective and high affinity agonists, antagonists, and radioligands for the study of P2Y1 receptors. Several of these molecules are now commercially available and are key reagents used by many laboratories to elucidate mechanism in this field of research. The long-term goal of our research is to identify subtype-selective agonists and antagonists for each of the nucleotide-activated P2Y receptors and to apply these molecules to increase molecular, physiological, and pathophysiological understanding of P2Y-receptor-dependent signaling. We recently have made considerable progress in defining structure activity relationships at the uridine nucleotide- activated P2Y2, P2Y4, and P2Y6 receptors and at the nucleotide sugar-activated P2Y14 receptor. Included in this progress is promising new insight for development of selective, high affinity antagonists of these receptors. Our proposed research plan will expand on these discoveries in important new directions. In the first specific aim we will synthesize and characterize selective high affinity agonists and antagonists of the UTP-activated P2Y2 and P2Y4 receptors. In the second specific aim we will synthesize and characterize selective high affinity agonists and antagonists of the nucleotide sugar-activated P2Y14 receptor and agonists of the UDP-activated P2Y6 receptor. Completion of the research described here will provide much-needed molecular tools to selectively activate and block uridine nucleotide- and nucleotide sugar-activated P2Y receptors with high affinity and selectivity. PUBLIC HEALTH RELEVANCE The drug targets studied in this research fulfill key roles in human physiology and in many diseases that affect the cardiovascular system, lung, brain, and other tissues. The impact on human health of an FDA-approved antagonist (clopidogrel;Plavix) of one of the receptors in the class of signaling proteins under study in this research program has been enormous. We anticipate that furthering the basic understanding of other receptors in this group of important signaling proteins will lead to similarly important therapeutic agents.